Most cells shed so-called extracellular vesicles or exosomes consisting of proteins and nucleic acids enclosed in phospholipid bilayers. Exosomes derived from cancer cells can be isolated.

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1+ crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1+ crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired subcutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening.

Overactivation of Wnt-β-catenin signaling, including β-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a β-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/β-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IκB-NF-κB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1–ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-κB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-κB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1–ezrin–NF-κB pathway, that induces IGFBP-2 expression, has an important role in CRC progression.

When certain types of cancer spread, they seem to prefer particular organs in the body, a choosiness that led Stephen Paget to propose the “seed and soil” hypothesis. This hypothesis, now more than 100 years old, suggests that different organs are somehow more receptive to certain types of cancer, just as different soils seem to allow some seeds, but not others, to find purchase.

While this hypothesis is as expressive as ever, it still lacks detail. It doesn’t suggest what mechanisms might drive organ-specific metastasis, or organotropic metastasis. The hypothesis, however, is being taken farther by researchers based at Weill Cornell Medicine. These researchers suggest that the old seed-and-soil idea, which sounds as haphazard as the dispersal of seeds by uncultivated plants, could be updated to describe a process that is more directed.

Essentially, a tumor metastasis may proceed the way settlers cultivate new land. First, scouts and pioneers are dispatched to identify fertile spots and develop basic infrastructure. Then, once the ground is prepared, settlers establish new communities.

In this scenario, the scouts are tumor exosomes. These exosomes are released by tumors in the millions, and they carry samples of the tumors’ proteins and genetic content. They fuse preferentially with cells at specific locations, and they ensure that recipient organs are prepared to host the tumor cells they represent.

Most important, this updated view of organotropic metastasis includes a mechanism to explain how exosomes are directed to specific organs. The exosomes, it turns out, are outfitted with particular sets of integrins, proteins that serve as a kind of destination label.

Supportive findings appeared October 28 in the journal Nature, in an article entitled, “Tumour exosome integrins determine organotropic metastasis.” This article described how the Weill Cornell researchers, in collaboration with scientists from the Memorial Sloan Kettering Cancer center and the Spanish National Cancer Research Centre (CNIO), examined exosomes from mouse and human lung-, liver-, and brain-tropic tumor cells. These exosomes were seen to fuse preferentially with resident cells at their predicted destinations, namely, lung fibroblasts and epithelial cells, liver Kupffer cells, and brain endothelial cells.

“Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis,” wrote the authors. “Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively.”

In other words, the study demonstrated the importance of integrins in metastatic nesting by blocking specific integrins in tumors that metastasize to specific organs. For example, when integrins were blocked in breast cancer, metastasis to lungs was reduced. Similarly, when integrins were blocked in pancreatic cancer, metastasis to liver was reduced.

In addition, the study showed that a tumor could be “tricked” by changing the integrin destination code of its exosomes. For example, a tumor that would normally go to the bones could be directed to the lungs instead.

“The integrin-specific signature that we identified may have significant value clinically, serving as a prognostic indicator for metastasis to specific organ sites,” said senior author David Lyden, M.D., Ph.D., the Stavros S. Niarchos Professor in Pediatric Cardiology and a professor of pediatrics and of cell and developmental biology at Weill Cornell Medicine. “Instead of waiting for late-stage metastasis, we can now initiate preventative strategies at an earlier point of disease progression with the hope of preventing its spread. This really changes the treatment paradigm.”

Using CRISPR as a High-Throughput Cancer Screening and Modeling Tool

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Using CRISPR/Cas9, scientists created a new high-throughput screening tool for studying the development and progression of liver cancer in mice. [Ernesto del Aguila III, NHGRI]

A contingent of researchers from the UK, Germany, and Spain have recently developed a novel CRISPR/Cas9 system that they believe can be utilized as a multiplexed screening approach to study and model cancer development in mice. In the current study, the investigators directly mutated genes within adult mouse livers to elucidate their role in cancer development and progression—simultaneously uncovering the gene combinations that coordinate to cause liver cancer.

“We reasoned that, by targeting mutations directly to adult liver cells using CRISPR/Cas9, we could better study and understand the biology of this important cancer,” explained co-author Mathias Friedrich, Ph.D., research scientist at the Wellcome Trust Sanger Institute. “Other approaches to engineer mutations in mice, such as stem cell manipulation, are limited by the laborious process, the long time frames and large numbers of animals needed. And, our method better mimics important aspects of human cancer biology than many “classic” mouse models: as in most human cancers, the mutations occur in the adult and only affect a few cells”.

The findings from this study were published online recently in PNAS through an article entitled “CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.”

This new approach is rapid, scalable, and extremely efficient, allowing the researchers to examine an array of genes or large regions of the genome concurrently. Moreover, this methodology affords scientists the ability to distinguish between cancer driver mutations and passenger mutations—those that occur as side-effects of cancer development.

The research team developed a list of up to eighteen genes with known or unknown evidence for their importance in two forms of liver cancer. They then introduced the CRISPR/Cas9 molecules, targeting various combinations of these genes into mice, which subsequently developed liver or bile duct cancer within a few months.

“Our approach enables us to simultaneously target multiple putative genes in individual cells,” noted co-author Roland Rad, Ph.D., project leader at the Technical University of Munich and the German Cancer Research Center Heidelberg. “We can now rapidly and efficiently screen which genes are cancer-causing and which ones are not. And, we can study how genes work together to cause cancers—a crucial piece of the puzzle we must solve to understand and tackle the disease.”

The investigators were able to confirm that a set of DNA-binding proteins called ARID (AT-rich interactive domain), influence the organization of chromosomes and are important for liver cancer development. Furthermore, mutations in a second protein, TET2, were found to be causative in bile duct cancer: although TET2 has not been found to be mutated in human biliary cancers, the proteins that it interacts with have been, showing that the CRISPR/Cas9 method can identify human cancer genes that are not mutated, but whose function is disturbed by other events.

“The new tools of targeting genes in combination and inducing insertions or deletions in chromosomes change our ability to identify new cancer-causing genes and to understand their role in cancer,” stated senior group leader and co-author Allan Bradley, Ph.D., director emeritus from the Sanger Institute. “Our results show that this approach is feasible and productive in liver cancer; we will now continue to study our new findings and try to extend the approach to other cancer types.”

This CRISPR/Cas9 approach may also be favorable for an in-depth examination of genomic deserts —regions within the human genome that appear to be devoid of genes. Yet, recent data from the ENCODE Project suggests that deserts can be populated, if not by genes, then by DNA regulatory regions that influence the activity of genes.

“Liver cancer has many DNA alterations in regions lacking genes: we don’t know which of these might be important for the disease,” said Dr. Rad. “However, we could show that it is now possible to delete such regions to systematically determine their role in liver cancer development.”

CRISPR Used to Create Mouse Models of Cancer

When scientists study the genetics of cancer, they often breed mice strains that carry selected cancer-associated mutations. But cultivating such strains, usually via transgenesis or gene targeting in embryonic stem cells, is often time-consuming and expensive. Could there be a better way—a faster, cheaper way—to create mice strains that carry particular genetic flaws?

An alternative has been proposed by researchers from MIT. They have shown that the CRISPR gene editing system can introduce cancer-causing mutations into the livers of adult mice. The researchers anticipate that their method will allow for more rapid testing of any single genes or gene combinations that are suspected of being capable of initiating tumor formation in the liver.

“The sequencing of human tumors has revealed hundreds of oncogenes and tumor suppressor genes in different combinations. The flexibility of this technology, as delivery gets better in the future, will give you a way to pretty rapidly test those combinations,” said Phillip Sharp, Ph.D., a professor at MIT’s Koch Institute for Integrative Cancer Research.

Dr. Sharp was part of the MIT research team, which was led by Koch Institute director Tyler Jacks, Ph.D. Dr. Jacks noted that the CRISPR technique, which not only provides the ability to delete genes, but also to replace them with altered versions, “really opens up all sorts of new possibilities when you think about the kinds of genes that you would want to mutate in the future.” Both loss of function and gain of function, he explained, are possible.

The MIT researchers presented their results August 6 in Nature, in an article entitled, “CRISPR-mediated direct mutation of cancer genes in the mouse liver.” It described how cancer models were generated using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice.

“We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumor suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination,” wrote the authors. “CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53.”

Studies of such genetically engineered mice have yielded many important discoveries, but the process, which requires introducing mutations into embryonic stem cells, can take more than a year and costs hundreds of thousands of dollars. Using Cas enzymes targeted to cut snippets of p53 and Pten, the researchers were able to disrupt those two genes in about 3% of liver cells, enough to produce liver tumors within three months.

With traditional techniques, genetically engineering such models is “a very long process,” commented Dr. Jacks. “And the more genes you’re working with, the longer and more complicated it becomes.

The researchers also used CRISPR to create a mouse model with an oncogene called beta catenin, which makes cells more likely to become cancerous if additional mutations occur later on. To create this model, the researchers had to cut out the normal version of the gene and replace it with an overactive form, which was successful in about 0.5% of hepatocytes.

In the Nature article, the authors emphasized that simplified methods of testing the oncogenic properties of candidates in vivo are critical. In particular, they cited the need to somehow evaluate the thousands of candidate cancer genes that are being discovered through next-generation sequencing efforts.

Already looking forward to refining their method of generating cancer models, the authors suggested that it could attain greater sensitivity if CRISPR/Cas9-mediated mutagenesis could be performed on a “sensitized” background carrying constitutive or conditional mutations in a tumor suppressor gene such as p53. “More efficient delivery techniques, such as adenovirus or adeno-associated virus, more potent sgRNAs, and longer homologous recombination templates,” they wrote, “might also improve the overall efficiency of this method and expand the range of tissue that could be targeted.”

Bioinformatics beyond Genome Crunching

Flow Cytometry, Workflow Development, and Other Information Stores Can Become Treasure Troves If You Use the Right IT Tools and Services

Advances in bioinformatics are no longer limited to just crunching through genomic and exosomic data. Bioinformatics, a discipline at the interface between biotechnology and information technology, also has lessons for flow cytometry and experimental design, as well as database searches, for both internal and external content.

One company offering variations on traditional genome crunching is DNAnexus. With the advent of the $1,000 genome, researchers find themselves drowning in data. To analyze the terabytes of information, they must contract with an organization to provide the computing power, or they must perform the necessary server installation and maintenance work in house.